Most secreted and plasma membrane proteins are synthesized on membrane-bound ribosomes at the endoplasmic reticulum (ER), where they cross the ER membrane or become embedded in it. Ribosomes synthesizing this class of proteins are targeted to the ER by an evolutionarily conserved molecular machine that consists of the signal recognition particle (SRP) and the SRP receptor (SR). SRP binds to signal sequences as they emerge from the ribosome as part of the growing polypeptide chain. The SRP that is bound to this ribosome-nascent chain complex (RNC) then interacts with the SR to effect the joining of the ribosome to a membrane-bound protein translocation channel (translocon), through which the nascent polypeptide is moved into the ER lumen or integrated into the ER membrane. The proposed work aims to investigate how SRP successfully monitors the cellular pool of ribosomes for the presence of signal sequences to assure their faithful recognition. Additionally, the question of how SRP coordinates its activity with that of other ribosome- associated protein biogenesis factors in the crowded molecular neighborhood surrounding the ribosome exit tunnel will be addressed. In particular, the hypothesis posing that SRP (as perhaps other factors) actively cycles on and off ribosomes in a mechanism that is obligatorily coupled to the ribosome's elongation cycle will be investigated. Using novel single molecule techniques in combination with other biophysical techniques, the process will be monitored in real time and in the context of the complete targeting machinery. Ultimately, a molecular movie will be constructed depicting the dynamic interactions between all of the molecular players that co-translationally target RNCs to the membrane. Specifically, the project will i) determine how the SRP- RNC interaction is modulated by the ribosomal elongation state and vice versa; ii) determine the lifetime and timing of the SRP interaction with RNCs relative to other translation factors; iii) determine the contribution to the SRP-RNC interaction of nascent chain length, presence or absence of a signal sequence, and signal sequence position; iv) dissect the molecular determinants on SRP that affect SRP-RNC dynamics; v) determine the effects of SR and translocon on SRP-RNC dynamics, thereby reconstituting the entire targeting reaction; vi) probe SRP conformational dynamics throughout the targeting reaction, and vii) determine the interplay of SRP/RNC interaction with that of other transient RNC binding partners. In addressing these questions, we aim at a high-resolution mechanistic definition of the core components shared by all SRP/SR targeting systems exemplified by the bacterial machinery. This will provide the framework to understand additional structural and regulatory complexities such as those found in higher eukaryotes. Ultimately, a precise molecular understanding of this highly conserved and ubiquitous protein biogenesis pathway- responsible for the proper biogenesis of virtually every signaling protein with which the cell communicates with its environment-is of profound significance to our understanding of cell physiology and pathology at a most fundamental level. PUBLIC HEALTH RELEVANCE: The vast majority of all secreted proteins and plasma membrane proteins (including virtually every growth factor receptor and its ligands) require co-translational targeting by SRP and SRP receptor for their proper biogenesis. The fidelity of this process is of immense importance, as the cell relies on the proper function of these signaling proteins for cues from the environment that ultimately determine its behavior, including such important decisions as when a cell should divide, differentiate, migrate, or die. A molecular understanding of how SRP and SR function is therefore of profound significance to our understanding of cell physiology and pathology at a most fundamental level, including that of many diseases.